CN106520798A - Identification and application of cotton drought-resistance related gene GhDRP1 - Google Patents
Identification and application of cotton drought-resistance related gene GhDRP1 Download PDFInfo
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- CN106520798A CN106520798A CN201611065602.0A CN201611065602A CN106520798A CN 106520798 A CN106520798 A CN 106520798A CN 201611065602 A CN201611065602 A CN 201611065602A CN 106520798 A CN106520798 A CN 106520798A
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Abstract
本发明公开了棉花抗旱相关基因GhDRP1鉴定及应用。一个新的棉花PP2C蛋白磷酸酶家族的基因GhDRP1，该基因在棉花干旱胁迫时表达受到诱导。GhDRP1的基因序列包含3个内含子和4个外显子。GhDRP1的基因编码区长1254bp，编码一个含有417氨基酸的蛋白质。GhDRP1蛋白定位于细胞核中。棉花受到干旱胁迫后，该基因表达显著上升，表明该基因是干旱诱导基因。实验表明，过量表达GhDRP1减弱了棉花的抗旱性，而抑制GhDRP1表达能够提高棉花的抗旱性。综上所述，本发明提供了一个在改良棉花抗旱性中具有重要应用价值的功能基因。The invention discloses the identification and application of the cotton drought resistance related gene GhDRP1. A new cotton PP2C protein phosphatase family gene, GhDRP1, whose expression is induced under drought stress in cotton. The gene sequence of GhDRP1 contains 3 introns and 4 exons. The gene coding region of GhDRP1 is 1254bp long, encoding a protein containing 417 amino acids. GhDRP1 protein is localized in the nucleus. After cotton was subjected to drought stress, the expression of this gene increased significantly, indicating that this gene is a drought-induced gene. Experiments showed that overexpressing GhDRP1 weakened the drought resistance of cotton, while inhibiting the expression of GhDRP1 could improve the drought resistance of cotton. In summary, the present invention provides a functional gene with important application value in improving the drought resistance of cotton.
Description
技术领域：Technical field:
本发明涉及一个棉花抗旱相关基因GhDRP1的克隆与功能鉴定。The invention relates to the cloning and functional identification of a cotton drought resistance related gene GhDRP1.
背景技术：Background technique:
植物生长发育与自然环境有密切的关系。干旱、洪涝、炎热、霜冻、盐碱、病虫害、杂草等都会对植物生长发育造成不利的影响，往往严重影响农作物(如水稻、小麦、棉花等)的产量和品质，有数据显示50％的作物产量降低都是由非生物胁迫引起的。水对植物的生命活动起着巨大的作用，任何溶质都要溶解在水中才能被吸收利用，据估计，全球因不良环境因素导致的作物减产的一半以上是干旱缺水所致。Plant growth and development are closely related to the natural environment. Drought, floods, heat, frost, salinity, pests, weeds, etc. will have adverse effects on plant growth and development, often seriously affecting the yield and quality of crops (such as rice, wheat, cotton, etc.), and some data show that 50% of the The reduction in crop yield is all caused by abiotic stress. Water plays a huge role in the life activities of plants, and any solute must be dissolved in water before it can be absorbed and utilized. It is estimated that more than half of the crop production reduction caused by adverse environmental factors in the world is caused by drought and water shortage.
棉花是世界上最主要的纤维作物，隶属锦葵科(Malvaceae)棉属(Gossypium)，原产于亚热带。依照其基因组构成可分为异源四倍体棉花(AADD)和二倍体棉花(AA或DD)。目前种植的主要是异源四倍体棉花(约占全部棉花种植面积的95％以上)，二倍体棉花种植较少。异源四倍体棉花又分为陆地棉(G.hirsutum)与海岛棉(G.barbadense)。陆地棉原产中美洲，其适应性广，是目前种植最多的棉花品种，约占全部棉花种植面积的90％以上。我国新疆及黄河长江流域，夏秋季常常晴热少雨，伏旱的发生比较频繁。棉花在此天气状况下，因蒸腾作用加大，水分供需失调，叶片出现萎蔫，棉株生长发育受阻，造成蕾铃加剧脱落现象，对棉花产量和品质影响很大。因此，培育耐旱棉花品种，对于我国棉花产业的长远发展极其重要。Cotton is the most important fiber crop in the world. It belongs to the Malvaceae genus Gossypium and is native to the subtropical zone. According to its genome composition, it can be divided into allotetraploid cotton (AADD) and diploid cotton (AA or DD). At present, allotetraploid cotton is mainly planted (accounting for more than 95% of the total cotton planting area), and diploid cotton is less planted. Allotetraploid cotton is further divided into G. hirsutum and G. barbadense. Upland cotton is originally produced in Central America and has wide adaptability. It is currently the most widely planted cotton variety, accounting for more than 90% of the total cotton planting area. In Xinjiang and the Yellow River and Yangtze River basins in my country, summer and autumn are often sunny and hot with little rain, and summer droughts occur frequently. Under the weather conditions of cotton, due to the increased transpiration, the imbalance of water supply and demand, the leaves will wilt, the growth and development of cotton plants will be hindered, and the phenomenon of bud shedding will be aggravated, which will have a great impact on cotton yield and quality. Therefore, cultivating drought-tolerant cotton varieties is extremely important for the long-term development of my country's cotton industry.
植物在长期进化过程中形成了一系列的信号转导机制来避免引起细胞内水分亏缺。已有研究表明，植物激素脱落酸(ABA)参与了包括种子萌发、休眠、营养器官发育以及胁迫耐受性等多种生理功能。其中，蛋白质的磷酸化和去磷酸化在ABA参与的信号转导途径中发挥重要功能。目前在植物中已鉴定了多种蛋白激酶和蛋白磷酸酶。有关蛋白激酶方面的研究较多，而蛋白磷酸酶方面的研究相对较少，已有的研究发现，与蛋白激酶一样，蛋白磷酸酶对生物体内的有关代谢也起着重要的调节作用。Plants have formed a series of signal transduction mechanisms in the long-term evolution process to avoid causing intracellular water deficit. Studies have shown that the plant hormone abscisic acid (ABA) is involved in various physiological functions including seed germination, dormancy, vegetative organ development, and stress tolerance. Among them, the phosphorylation and dephosphorylation of proteins play an important role in the signal transduction pathway in which ABA participates. A variety of protein kinases and protein phosphatases have been identified in plants. There are many studies on protein kinases, but relatively few studies on protein phosphatases. Existing studies have found that, like protein kinases, protein phosphatases also play an important role in regulating the metabolism in vivo.
根据底物特异性，真核生物中蛋白磷酸酶可分为两大类：酪氨酸蛋白磷酸酶(PTP)和丝氨酸或苏氨酸蛋白磷酸酶。酪氨酸蛋白磷酸酶又分为酪氨酸蛋白磷酸酶(PTP)和双特异性蛋白磷酸酶(DSPTP)。根据氨基酸序列和晶体结构的不同，丝氨酸/苏氨酸蛋白磷酸酶的分类为PPP和PPM的基因家族。PPP家族蛋白磷酸酶又分为3类：PP1，PP2A和PP2B，而PPM家族蛋白磷酸酶包括PP2C和丙酮酸脱氢酶磷酸酶。研究表明植物中PP2C可能在许多信号转导途径中都作为负调控因子起作用，调节各种环境胁迫(如干旱，冷，伤害等)。虽然目前对PP2C的研究取得了较大的进展，但对于植物PP2C的功能及其作用底物仍知之甚少。According to substrate specificity, protein phosphatases in eukaryotes can be divided into two major categories: tyrosine protein phosphatases (PTPs) and serine or threonine protein phosphatases. Tyrosine protein phosphatases are further divided into tyrosine protein phosphatases (PTPs) and dual-specificity protein phosphatases (DSPTPs). According to the differences in amino acid sequence and crystal structure, serine/threonine protein phosphatases are classified into PPP and PPM gene families. The PPP family of protein phosphatases is further divided into three categories: PP1, PP2A, and PP2B, while the PPM family of protein phosphatases includes PP2C and pyruvate dehydrogenase phosphatases. Studies have shown that PP2C in plants may act as a negative regulator in many signal transduction pathways, regulating various environmental stresses (such as drought, cold, injury, etc.). Although the current research on PP2C has made great progress, the function and substrate of plant PP2C are still poorly understood.
PP2C是一类含丝氨酸/苏氨酸残基的蛋白磷酸酶，酶催化活性依赖于Mg2+或Mn2+。PP2C蛋白磷酸酶没有调节亚基，是一种单体酶，酶的催化区域有11个保守的基序，这个基序与细胞内的信号转导有关，其中包括一些跨膜区域和一些参与蛋白激酶互作的区域。真核生物中PP2C的催化结构域在蛋白N端或C端。拟南芥76个PP2C中，有44个PP2C的催化区域是位于C端，其余的则位于N端。除了6个PP2C不能聚类以外，其他拟南芥PP2C可分为10类(A–J)。PP2C蛋白磷酸酶通过去磷酸化作用负调控蛋白激酶级联信号系统，参与细胞周期、胁迫信号转导、基因转录、蛋白质翻译及翻译后修饰等细胞活动过程。目前研究最多的是A类PP2C，发现它们在ABA信号通路中起重要作用，与SnRKs构成ABA信号通路的核心。当植物受干旱或高盐胁迫时，体内的ABA积累，ABA与受体PYL结合后，促进PYL与PP2C结合，形成ABA-PYL-PP2C三元复合体，这样就使得被PP2C去磷酸化而失活的SnRKs重新被磷酸化而激活，被激活的SnRKs磷酸化其下游的钾离子通道蛋白和ABF/bZIP蛋白，从而调节ABA诱导基因的表达，进而调节气孔的开闭，种子的萌发等。对拟南芥PP2C单突变体、双突变体以及多突变体的研究表明，当PP2C功能缺失后植物提高了抗旱能力。在植物体中PYL的量是有限的，而PP2C的表达会受ABA的诱导。因此，过量的PP2C会进一步抑制SnRKs的活性。ABA对种子萌发和休眠、叶片的气孔关闭和水分控制等都有显著的调控作用，也能调节植物对外界环境胁迫的响应。大量研究表明PP2C与AB A信号途径有关。拟南芥abi l和abi2突变体对ABA非常敏感，这表明ABI1基因丧失功能后植物对ABA的敏感性增强，ABI l是ABA信号途径的负调控因子。Merlot等筛选出abi2的隐性回复突变体abi 2-1R，其PP2C酶活性比野生型ABI2低100倍，但在种子萌发与调节气孔关闭上均与野生型无异。而双突变体(abi 1-1R和abi 1-1R)则又在种子萌发上比任何一个单突变体对ABA更敏感，于是认为ABI2也是ABA信号途径的负调控因子。ABI2与ABIl编码的蛋白质氨基酸序列有较高的同源性，二者包含PP2C核心区域的C-端区域有86％同源，不保守的N-端区域也有48％的同源性，但ABI1的N-端延伸区包含一个Ca 2+结合位点的EF手型结构域，而ABI2的N-端延伸区不含EF-手型结构域，说明二者可能存在功能冗余。拟南芥PP2CA主要在叶中大量表达，在原生质体中瞬时表达能够阻断ABA信号途径。PP2CA的转录可受低温、干旱、高盐和ABA诱导，低温处理ABA缺陷型突变体aba1-1后PP2CA的转录降低，说明低温诱导的该基因表达是依赖ABA的，而干旱诱导的PP2CA基因表达是依赖ABA和ABI的。抑制PP2CA基因表达加速植物的冷适应性，从而提高其抗冻性。实验表明，PP2CA是ABA信号途径的负调控因子。此外，PP2CA能够与控制K+内向通道的蛋白AKT 2相互作用，推测PP2CA可能通过与AKT2相互作用从而调控K+内向通道。PP2C is a class of protein phosphatases containing serine/threonine residues, and its catalytic activity depends on Mg 2+ or Mn 2+ . PP2C protein phosphatase has no regulatory subunits and is a monomeric enzyme. The catalytic region of the enzyme has 11 conserved motifs, which are related to signal transduction in cells, including some transmembrane regions and some participating proteins Kinase interaction domain. The catalytic domain of PP2C in eukaryotes is at the N- or C-terminus of the protein. Among the 76 PP2Cs in Arabidopsis thaliana, the catalytic regions of 44 PP2Cs are located at the C-terminus, and the rest are located at the N-terminus. Except for 6 PP2Cs that could not be clustered, other Arabidopsis PP2Cs could be classified into 10 groups (A–J). PP2C protein phosphatase negatively regulates the protein kinase cascade signaling system through dephosphorylation, and participates in cell cycle, stress signal transduction, gene transcription, protein translation and post-translational modification and other cellular activities. At present, the most studied ones are class A PP2Cs, and it is found that they play an important role in the ABA signaling pathway, and form the core of the ABA signaling pathway with SnRKs. When plants are under drought or high-salt stress, ABA in the body accumulates. After ABA binds to the receptor PYL, it promotes the combination of PYL and PP2C to form an ABA-PYL-PP2C ternary complex, which makes it dephosphorylated by PP2C and loses The active SnRKs are reactivated by phosphorylation, and the activated SnRKs phosphorylate their downstream potassium ion channel proteins and ABF/bZIP proteins, thereby regulating the expression of ABA-induced genes, and then regulating the opening and closing of stomata, seed germination, etc. Studies on Arabidopsis PP2C single mutants, double mutants and multiple mutants have shown that plants have improved drought resistance when PP2C function is lost. The amount of PYL in plants is limited, while the expression of PP2C is induced by ABA. Therefore, excess PP2C would further inhibit the activity of SnRKs. ABA has significant regulatory effects on seed germination and dormancy, leaf stomatal closure and water control, and can also regulate plant responses to external environmental stress. A large number of studies have shown that PP2C is related to the ABA signaling pathway. Arabidopsis abi l and abi2 mutants are very sensitive to ABA, which indicates that plants are more sensitive to ABA after the loss of function of ABI1 gene, and ABI l is a negative regulator of ABA signaling pathway. Merlot et al. screened out the recessive back mutant abi2-1R of abi2. Its PP2C enzyme activity was 100 times lower than that of the wild-type ABI2, but it was no different from the wild-type in terms of seed germination and regulation of stomatal closure. The double mutants (abi 1-1R and abi 1-1R) are more sensitive to ABA than any single mutant in seed germination, so ABI2 is considered to be a negative regulator of ABA signaling pathway. The amino acid sequence of the protein encoded by ABI2 and ABI1 has high homology, the C-terminal region of the two containing the core region of PP2C has 86% homology, and the non-conserved N-terminal region also has 48% homology, but ABI1 The N-terminal extension of ABI2 contains an EF-hand domain with a Ca 2+ binding site, but the N-terminal extension of ABI2 does not contain an EF-hand domain, indicating that there may be functional redundancy between the two. Arabidopsis PP2CA is mainly expressed in large quantities in leaves, and transient expression in protoplasts can block the ABA signaling pathway. The transcription of PP2CA can be induced by low temperature, drought, high salt and ABA, and the transcription of PP2CA decreased after low temperature treatment of the ABA-deficient mutant aba1-1, indicating that the gene expression induced by low temperature is dependent on ABA, while the expression of PP2CA gene induced by drought It is dependent on ABA and ABI. Inhibition of PP2CA gene expression accelerates the cold adaptation of plants, thereby improving their frost resistance. Experiments have shown that PP2CA is a negative regulator of the ABA signaling pathway. In addition, PP2CA can interact with the protein AKT 2 that controls the K + inward channel. It is speculated that PP2CA may regulate the K + inward channel by interacting with AKT2.
作为植物体内较大的一类蛋白磷酸酶家族，PP2C具有特殊的结构特征和理化性质，并参与植物体内多种信号途径，而植物体内PP2C的多样性则表明其在不同的组织和器官中信号转导机制的多样性。因此，深入研究PP2C的生物学功能及作用机制，具有重要理论意义和应用价值。特别是棉花中关于PP2C蛋白磷酸酶家族的研究报导极少，因此，对于PP2C在棉花抗旱中的功能研究，将有助于揭示棉花抗旱应答的分子调控机制，克隆鉴定棉花抗旱相关的重要基因，直接应用于棉花品种改良。As a large family of protein phosphatases in plants, PP2C has special structural characteristics and physical and chemical properties, and participates in various signaling pathways in plants, and the diversity of PP2C in plants indicates that it signals in different tissues and organs. Diversity of transduction mechanisms. Therefore, in-depth study of the biological function and mechanism of PP2C has important theoretical significance and application value. In particular, there are very few reports on the PP2C protein phosphatase family in cotton. Therefore, research on the function of PP2C in cotton drought resistance will help to reveal the molecular regulation mechanism of cotton drought resistance response, clone and identify important genes related to cotton drought resistance, It is directly applied to the improvement of cotton varieties.
发明内容Contents of the invention
本发明的目的在于提供一个新的棉花抗逆相关的PP2C基因(GhDRP1)，分析揭示该基因的生物学功能，探索其在棉花抗逆过程中的作用机制，进而应用该基因改良棉花的抗旱性，创造棉花抗旱优良品种。The purpose of the present invention is to provide a new PP2C gene (GhDRP1) related to cotton stress resistance, analyze and reveal the biological function of the gene, explore its mechanism of action in the process of cotton stress resistance, and then apply the gene to improve the drought resistance of cotton , to create excellent drought-resistant varieties of cotton.
为了研究棉花抗旱的分子机制，我们对两个不同品种(晋棉13号和鲁棉6号)的棉花幼苗进行了干旱处理，分别收取了正常浇水条件和干旱处理3天、5天和7天的棉花叶片材料，提取RNA，委托武汉生命之美公司进行转录组测序分析。分析结果表明，GhDRP1基因的表达是受干旱诱导的。在较短时间干旱处理时，该基因在鲁棉6号叶片中诱导表达水平高于晋棉13号，但在长时间干旱条件下，该基因诱导表达水平在晋棉13号叶片中要高于鲁棉6号(如图2所示)。序列分析发现，该基因编码一个含有PP2C结构域的蛋白质，序列如SEQ.ID.No.3所示，其与拟南芥A类PP2C蛋白的同源性较高，因此推测GhDRP1可能具有拟南芥A类PP2C相似的功能。In order to study the molecular mechanism of drought resistance in cotton, we subjected cotton seedlings of two different varieties (Jinmian 13 and Lumian 6) to drought treatment, and received normal watering conditions and drought treatment for 3 days, 5 days and 7 days Tian’s cotton leaf materials were used to extract RNA, and entrusted Wuhan Beauty of Life Company to conduct transcriptome sequencing analysis. The analysis results showed that the expression of GhDRP1 gene was induced by drought. Under shorter drought conditions, the induced expression level of this gene in the leaves of Lumian No. 6 was higher than that of Jinmian No. 13, but under long-term drought conditions, the induced expression level of this gene was higher in the leaves of Jinmian No. 13 Lumian No. 6 (as shown in Figure 2). Sequence analysis found that this gene encodes a protein containing a PP2C domain, the sequence is shown in SEQ.ID.No.3, which has a high homology with the Arabidopsis A class PP2C protein, so it is speculated that GhDRP1 may have Arabidopsis Mustard A class PP2C has a similar function.
为了研究该蛋白的亚细胞定位，构建了P2300-GFP-GhDRP1的载体，将该载体转化拟南芥，获得转基因植株。在共聚焦显微镜下观察转基因拟南芥幼苗根部细胞的GFP荧光，结果显示GhDRP1定位在细胞核中(如图3所示)。由于GhDRP1的全长蛋白具有转录激活活性，因此将编码GhDRP1蛋白的开放阅读框序列截成4段：GhDRP1(1-354)、GhDRP1(355-1050)、GhDRP1(355-1134)及GhDRP1(355-1254)，然后构建这4段截短形式的DNA序列的酵母表达载体，分别分析其编码蛋白的转录激活活性，结果显示这4种截短形式的蛋白都不具有转录激活活性(如图4所示)。In order to study the subcellular localization of the protein, a P2300-GFP-GhDRP1 vector was constructed, and the vector was transformed into Arabidopsis thaliana to obtain transgenic plants. The GFP fluorescence of the root cells of the transgenic Arabidopsis seedlings was observed under a confocal microscope, and the results showed that GhDRP1 was localized in the nucleus (as shown in FIG. 3 ). Since the full-length protein of GhDRP1 has transcription activation activity, the open reading frame sequence encoding GhDRP1 protein was truncated into four segments: GhDRP1(1-354), GhDRP1(355-1050), GhDRP1(355-1134) and GhDRP1(355 -1254), and then construct the yeast expression vectors of these 4 truncated DNA sequences, and analyze the transcriptional activation activity of their encoded proteins respectively. shown).
为研究GhDRP1的功能，构建了GhDRP1过量表达载体，转化拟南芥，获得转基因拟南芥植株。在ABA和甘露醇处理下，转基因拟南芥种子的萌发速率和绿苗率要高于野生型，这表明GhDRP1过量表达增强了拟南芥种子对ABA和甘露醇的耐受性(如图5所示)。In order to study the function of GhDRP1, an overexpression vector of GhDRP1 was constructed and transformed into Arabidopsis thaliana to obtain transgenic Arabidopsis plants. Under the treatment of ABA and mannitol, the germination rate and green shoot rate of transgenic Arabidopsis seeds were higher than those of wild type, which indicated that the overexpression of GhDRP1 enhanced the tolerance of Arabidopsis seeds to ABA and mannitol (Fig. 5 shown).
构建了GhDRP1过量表达(OE)和RNA干扰(RNAi)载体，分别转化棉花，获得转基因棉花植株。提取GhDRP1OE和GhDRP1RNAi转基因棉花叶片RNA，反转录成cDNA，检测在转基因棉花中GhDRP1基因表达水平。结果表明，转基因棉花叶片中GhDRP1表达水平都有不同程度的上调和下调(如图6所示)。对T1代转基因棉花进行干旱处理，结果显示在干旱处理后GhDRP1OE植株中丙二醛(MDA)和双氧水(H2O2)含量显著高于对照品种晋棉13号(J13)，而GhDRP1RNAi转基因棉花植株中MDA和H2O2含量显著低于对照品种。相反，GhDRP1RNAi植株中超氧化物歧化酶(SOD)和过氧化物酶(POD)活性显著升高，而GhDRP1过量表达植株中SOD和POD酶活性显著降低。这些结果表明过量表达GhDRP1减弱了棉花的抗旱性，而抑制GhDRP1基因表达能够提高棉花的抗旱性。GhDRP1 overexpression (OE) and RNA interference (RNAi) vectors were constructed, and transformed into cotton respectively to obtain transgenic cotton plants. Extract GhDRP1OE and GhDRP1RNAi transgenic cotton leaf RNA, reverse transcribe into cDNA, and detect the expression level of GhDRP1 gene in transgenic cotton. The results showed that the expression levels of GhDRP1 in the transgenic cotton leaves were up-regulated and down-regulated to varying degrees (as shown in FIG. 6 ). Drought treatment was carried out on T1 transgenic cotton, and the results showed that the contents of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) in GhDRP1OE plants were significantly higher than those in the control variety Jinmian 13 (J13) after drought treatment, while GhDRP1RNAi transgenic cotton The contents of MDA and H 2 O 2 in the plants were significantly lower than those in the control varieties. In contrast, the activities of superoxide dismutase (SOD) and peroxidase (POD) were significantly increased in GhDRP1 RNAi plants, while the activities of SOD and POD enzymes were significantly decreased in GhDRP1 overexpression plants. These results indicated that overexpression of GhDRP1 weakened the drought resistance of cotton, while suppressing GhDRP1 gene expression could improve the drought resistance of cotton.
本发明的优点：Advantages of the present invention:
1.提供了一个新的受干旱诱导表达的GhDRP1基因序列及cDNA序列。该基因在正常条件下的棉花叶片中的表达量较低，但在干旱处理条件下，该基因的表达受到诱导而显著升高，表明该基因可能在棉花应答干旱胁迫中起重要作用。1. Provide a new GhDRP1 gene sequence and cDNA sequence induced by drought. The expression level of this gene in cotton leaves under normal conditions was low, but under drought treatment conditions, the expression of this gene was induced and increased significantly, indicating that this gene may play an important role in the response of cotton to drought stress.
2.GhDRP1基因编码一个PP2C蛋白磷酸酶，该蛋白定位于细胞核中。无论在有或无ABA的情况下，该蛋白都能够与GhPYL蛋白发生相互作用，且GhDRP1转基因拟南芥对ABA不敏感，说明该蛋白调控棉花应答抗旱胁迫可能通过两种不同的信号途径：依赖于ABA的信号通路和不依赖于ABA的信号通路。2. GhDRP1 gene encodes a PP2C protein phosphatase, which localizes in the nucleus. This protein can interact with GhPYL protein in the presence or absence of ABA, and GhDRP1 transgenic Arabidopsis is not sensitive to ABA, indicating that this protein may regulate cotton response to drought stress through two different signaling pathways: dependent on ABA-dependent signaling pathways and ABA-independent signaling pathways.
3.在干旱处理条件下，GhDRP1过量表达转基因棉花植株中丙二醛(MDA)和双氧水(H2O2)含量显著高于对照品种晋棉13号，而GhDRP1RNAi转基因棉花植株中MDA和H2O2含量显著低于对照品种。相反，GhDRP1RNAi植株中超氧化物歧化酶(SOD)和过氧化物酶(POD)活性显著升高，而GhDRP1过量表达植株中SOD和POD酶活性显著降低。实验表明，过量表达GhDRP1减弱了棉花的抗旱性，而抑制GhDRP1表达能够提高棉花的抗旱性。3. Under drought treatment, the contents of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) in GhDRP1 overexpressed transgenic cotton plants were significantly higher than those in the control variety Jinmian 13, while the contents of MDA and H 2 O 2 in GhDRP1RNAi transgenic cotton plants The O2 content was significantly lower than that of the control variety. In contrast, the activities of superoxide dismutase (SOD) and peroxidase (POD) were significantly increased in GhDRP1 RNAi plants, while the activities of SOD and POD enzymes were significantly decreased in GhDRP1 overexpression plants. Experiments showed that overexpressing GhDRP1 weakened the drought resistance of cotton, while inhibiting the expression of GhDRP1 could improve the drought resistance of cotton.
本发明通过以下附图和实施作进一步阐述，但不限制本发明的范围。The present invention is further illustrated by the following drawings and implementations, but does not limit the scope of the present invention.
附图说明：Description of drawings:
图1.荧光定量RT-PCR分析GhDRP1在棉花各组织中的表达。R，根；H，下胚轴；C，子叶；T，真叶；P，花瓣；A，花药；O，开花当天胚珠；F，开花后9天纤维。Figure 1. Fluorescent quantitative RT-PCR analysis of the expression of GhDRP1 in various tissues of cotton. R, root; H, hypocotyl; C, cotyledon; T, true leaf; P, petal; A, anther; O, ovule on the day of anthesis; F, fiber 9 days after anthesis.
图2.荧光定量RT-PCR分析在干旱处理条件下GhDRP1在棉花中的表达。J13:晋棉13号；Lu6:鲁棉6号；CK:对照(未经干旱处理)；3d，5d，7d:干旱处理3,5和7天。Figure 2. Fluorescent quantitative RT-PCR analysis of GhDRP1 expression in cotton under drought treatment conditions. J13: Jinmian 13; Lu6: Lumian 6; CK: control (without drought treatment); 3d, 5d, 7d: 3, 5 and 7 days of drought treatment.
图3.GhDRP1蛋白在拟南芥细胞中的亚细胞定位。将GhDRP1:eGFP载体转化拟南芥，用共聚焦显微镜观察转基因拟南芥植株根部细胞的GFP荧光，发现荧光信号主要分布在细胞核中。A图是明视野下的拟南芥植株根部细胞图像；B图是暗视野下的GFP荧光图像；C图是A和B图重叠后的图像。Figure 3. Subcellular localization of GhDRP1 protein in Arabidopsis cells. The GhDRP1:eGFP vector was transformed into Arabidopsis thaliana, and the GFP fluorescence of the root cells of the transgenic Arabidopsis plants was observed with a confocal microscope, and it was found that the fluorescent signal was mainly distributed in the nucleus. Figure A is the root cell image of Arabidopsis thaliana plant under bright field; Figure B is the GFP fluorescence image under dark field; Figure C is the overlapping image of A and B.
图4.GhDRP1蛋白转录激活功能检测。Figure 4. GhDRP1 protein transcriptional activation function detection.
将pGBKT7-GhDRP1、pGBKT7-GhDRP1(1-354)、pGBKT7-GhDRP1(355-1050)、pGBKT7-GhDRP1(355-1134)和pGBKT7-GhDRP1(355-1254)载体分别转化酵母细胞Y187,将检测得到的阳性酵母菌落在SD/-Ade/-Trp选择培养基上划线，能够生长的为阳性，说明GhDRP1蛋白具有转录激活活性，否则，则说明没有转录激活活性。同时，将阳性酵母菌落进行X-gal显色，显蓝色的为阳性，说明GhDRP1蛋白具有转录激活功能，而不显蓝色的为阴性，说明不具有转录激活功能。The pGBKT7-GhDRP1, pGBKT7-GhDRP1(1-354), pGBKT7-GhDRP1(355-1050), pGBKT7-GhDRP1(355-1134) and pGBKT7-GhDRP1(355-1254) vectors were respectively transformed into yeast cell Y187, and the obtained The positive yeast colonies were streaked on the SD/-Ade/-Trp selection medium, and those that could grow were positive, indicating that the GhDRP1 protein had transcriptional activation activity, otherwise, it indicated that there was no transcriptional activation activity. At the same time, the positive yeast colonies were subjected to X-gal color development, and the blue color was positive, indicating that the GhDRP1 protein had a transcription activation function, while the non-blue color was negative, indicating that it did not have a transcription activation function.
图5.ABA和甘露醇对GhDRP1转基因拟南芥种子萌发率和绿苗率的影响。Figure 5. Effects of ABA and mannitol on the germination rate and green shoot rate of GhDRP1 transgenic Arabidopsis thaliana seeds.
A-C图.GhDRP1过量表达拟南芥在含有1μM ABA的MS培养基上生长情况(图A)及在正常条件(CK)和ABA存在的条件下种子萌发率(图B)和绿苗率(图C)的统计分析结果；D-F图.GhDRP1过量表达拟南芥在含有300mM甘露醇(mannitol)的MS培养基上生长的情况(图D)及在正常条件(CK)和甘露醇存在的条件下种子萌发率(图E)和绿苗率(图F)的统计分析结果。结果显示GhDRP1过量表达拟南芥对ABA不敏感，种子萌发率和绿苗率均高于野生型(WT)；而GhDRP1过量表达拟南芥对甘露醇敏感，种子萌发率和绿苗率均低于野生型(WT)。WT：野生型；L9–L12：转基因拟南芥株系。1d–8d：分别代表种子萌发的天数(1–8天)。Figures A-C. GhDRP1 overexpressed Arabidopsis growth on MS medium containing 1 μM ABA (Figure A) and seed germination rate (Figure B) and green shoot rate (Figure B) under normal conditions (CK) and the presence of ABA C) Statistical analysis results; D-F diagrams. GhDRP1 overexpression Arabidopsis growth on MS medium containing 300mM mannitol (figure D) and under normal conditions (CK) and mannitol presence conditions Statistical analysis results of seed germination rate (Figure E) and green shoot rate (Figure F). The results showed that GhDRP1 overexpressed Arabidopsis was not sensitive to ABA, and the seed germination rate and green shoot rate were higher than those of wild type (WT); while GhDRP1 overexpressed Arabidopsis was sensitive to mannitol, and the seed germination rate and green shoot rate were lower in wild type (WT). WT: wild type; L9–L12: transgenic Arabidopsis lines. 1d–8d: represent the days of seed germination (1–8 days), respectively.
图6.干旱条件下GhDRP1过量表达拟南芥植株表型分析。Figure 6. Phenotype analysis of Arabidopsis plants overexpressed with GhDRP1 under drought conditions.
A图.正常条件下生长的WT和转基因植株的生长情况；B图.干旱处理20天后WT和转基因植株的生长情况；C图.干旱处理23天后复水1天后WT和转基因植株的生长情况；D图.复水2天后WT和转基因植株的存活率。结果显示GhDRP1过量表达拟南芥植株相对于野生型对干旱更加敏感。WT：野生型；L9–L12：转基因拟南芥株系；CK：植株在正常条件下生长；Drought：植株在干旱条件下生长。Figure A. The growth of WT and transgenic plants grown under normal conditions; Figure B. The growth of WT and transgenic plants after 20 days of drought treatment; Figure C. The growth of WT and transgenic plants after 23 days of drought treatment and rehydration for 1 day; Panel D. Survival rates of WT and transgenic plants 2 days after rehydration. The results showed that Arabidopsis plants overexpressing GhDRP1 were more sensitive to drought than wild-type plants. WT: wild type; L9–L12: transgenic Arabidopsis lines; CK: plants grown under normal conditions; Drought: plants grown under drought conditions.
图7.GhDRP1转基因棉花植株叶片生理指标测定。Fig. 7. Determination of physiological indexes of leaves of GhDRP1 transgenic cotton plants.
A图.荧光定量RT-PCR分析GhDRP1基因在野生型和转基因棉花叶片中的表达；B图.干旱处理条件下柯字312(WT)、晋棉13号(J13)、鲁棉6号(Lu6)及GhDRP1过量表达(OE)和RNAi转基因棉花植株叶片中丙二醛和双氧水含量及超氧化物歧化酶和过氧化物酶活性。POEL，过量表达转基因棉花株系；PRNAiL，RNAi转基因棉花株系。CK，未经干旱处理；Drought，干旱处理。A panel. Fluorescent quantitative RT-PCR analysis of GhDRP1 gene expression in wild-type and transgenic cotton leaves; B panel. Kezi 312 (WT), Jinmian 13 (J13), Lumian 6 (Lu6 ) and GhDRP1 overexpression (OE) and RNAi transgenic cotton plant leaf malondialdehyde and hydrogen peroxide content and superoxide dismutase and peroxidase activities. POEL, overexpressed transgenic cotton lines; PRNAiL, RNAi transgenic cotton lines. CK, no drought treatment; Drought, drought treatment.
在干旱处理条件下，GhDRP1过量表达转基因棉花植株中丙二醛(MDA)和双氧水(H2O2)含量显著高于对照品种，而GhDRP1RNAi转基因棉花植株中MDA和H2O2含量显著低于对照品种。相反，GhDRP1RNAi植株中超氧化物歧化酶(SOD)和过氧化物酶(POD)酶活性显著升高，而GhDRP1过量表达植株中SOD和POD酶活性显著降低。Under drought treatment, the contents of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) in GhDRP1 overexpressed transgenic cotton plants were significantly higher than those in control varieties, while the contents of MDA and H 2 O 2 in GhDRP1 RNAi transgenic cotton plants were significantly lower control species. On the contrary, the enzyme activities of superoxide dismutase (SOD) and peroxidase (POD) were significantly increased in GhDRP1 RNAi plants, while the activities of SOD and POD enzymes were significantly decreased in GhDRP1 overexpression plants.
具体实施方式：detailed description:
一个棉花PP2C家族新基因GhDRP1全长序列的克隆鉴定和功能分析：Cloning identification and functional analysis of the full-length sequence of a new cotton PP2C family gene GhDRP1:
1.GhDRP1基因及cDNA序列的分离鉴定1. Isolation and identification of GhDRP1 gene and cDNA sequence
利用RNA-seq技术分析两个棉花品种(晋棉13号和鲁棉6号)的转录组，通过DNA和蛋白质序列比较分析，鉴定了一个干旱诱导表达的基因GhDRP1。然后，以晋棉13号棉花为材料，分离纯化叶片总RNA，进而分离获得GhDRP1全长cDNA序列，如SEQ ID NO.2所示，总长1254bp。按照GhDRP1cDNA序列设计引物，以棉花基因组DNA为模板，采用PCR技术扩增获得该基因的DNA全长序列，如SEQ ID NO.1所示，总长1873bp，其包含4个外显子和3个内含子(下划线)，第一内含子从496-602bp，共107bp，第二内含子从909-1329bp，共421bp，第三内含子从1436-1526bp，共91bp。Using RNA-seq technology to analyze the transcriptomes of two cotton varieties (Jinmian 13 and Lumian 6), a drought-induced gene GhDRP1 was identified through comparative analysis of DNA and protein sequences. Then, Jinmian No. 13 cotton was used as the material to isolate and purify the total RNA of leaves, and then isolate and obtain the full-length cDNA sequence of GhDRP1, as shown in SEQ ID NO.2, with a total length of 1254bp. Primers were designed according to the GhDRP1 cDNA sequence, and the cotton genomic DNA was used as a template to amplify the full-length DNA sequence of the gene by PCR technology. As shown in SEQ ID NO.1, the total length is 1873bp, which includes 4 exons and 3 introns Introns (underlined), the first intron is from 496-602bp, a total of 107bp, the second intron is from 909-1329bp, a total of 421bp, and the third intron is from 1436-1526bp, a total of 91bp.
2.定量RT-PCR分析GhDRP1基因表达2. Quantitative RT-PCR analysis of GhDRP1 gene expression
利用RNA提取试剂盒(Qiagin)提取纯化棉花不同组织和纤维的总RNA。实时荧光定量RT-PCR研究基因的表达按照Li XB,Fan XP,Wang XL,Cai L,Yang WC,2005.The CottonACTIN1gene is functionally expressed in fibers and participates in fiberelongation.Plant Cell17:859–875进行。Total RNA from different tissues and fibers of cotton was extracted and purified using RNA extraction kit (Qiagin). Real-time fluorescent quantitative RT-PCR to study gene expression was carried out according to Li XB, Fan XP, Wang XL, Cai L, Yang WC, 2005. The CottonACTIN1 gene is functionally expressed in fibers and participates in fiberrelongation. Plant Cell17:859–875.
首先，将棉花根、下胚轴、子叶、真叶、花瓣、花药、胚珠和纤维的总RNA(2μg/样)用M-MLV Reverse Transcriptase(Promega)反转录成cDNA；然后，以cDNA为模板，用基因特异引物(GhDRP1RTP1和GhDRP1RTP2)和Real-time PCR Master Mix(TOYOBO,Japan)进行定量PCR反应。棉花GhUBI1基因作为RT-PCR反应的内标，目标基因每一个循环的扩增都被SYBR-Green荧光检测，计算目标基因的表达水平相对值。每一实验重复3次，统计分析实验结果，见图1和图2。First, the total RNA (2 μg/sample) of cotton roots, hypocotyls, cotyledons, true leaves, petals, anthers, ovules and fibers was reverse-transcribed into cDNA with M-MLV Reverse Transcriptase (Promega); then, cDNA was used as Template, quantitative PCR reaction was performed with gene-specific primers (GhDRP1RTP1 and GhDRP1RTP2) and Real-time PCR Master Mix (TOYOBO, Japan). The cotton GhUBI1 gene was used as the internal standard of the RT-PCR reaction, and the amplification of each cycle of the target gene was detected by SYBR-Green fluorescence, and the relative expression level of the target gene was calculated. Each experiment was repeated 3 times, and the experimental results were statistically analyzed, as shown in Figure 1 and Figure 2.
3.GhDRP1蛋白亚细胞定位分析3. GhDRP1 protein subcellular localization analysis
构建p2300-N-eGFP-GhDRP1植物表达载体，将该载体通过电转化法转入农杆菌LBA4404中，通过浸花法转化拟南芥，收获T0代种子后筛选阳性植株，将筛选出的阳性拟南芥小苗置于共聚焦显微镜下，观察根部细胞的GFP荧光，见图3。图3发现荧光信号主要分布在细胞核中。The p2300-N-eGFP-GhDRP1 plant expression vector was constructed, and the vector was transformed into Agrobacterium LBA4404 by electroporation, Arabidopsis was transformed by flower dipping method, positive plants were screened after T0 generation seeds were harvested, and the screened positive plants were screened. The Arabidopsis thaliana seedlings were placed under a confocal microscope to observe the GFP fluorescence of the root cells, as shown in Figure 3. Figure 3 found that the fluorescent signal was mainly distributed in the nucleus.
4.GhDRP1蛋白激活功能的分析4. Analysis of GhDRP1 protein activation function
构建pGBKT7-GhDRP1、pGBKT7-GhDRP1(1-354)、pGBKT7-GhDRP1(355-1050)、pGBKT7-GhDRP1(355-1134)和pGBKT7-GhDRP1(355-1254)载体，分别转化酵母细胞Y187,将检测得到的阳性酵母菌落在SD/-Ade/-Trp培养基上划线，如果能够生长则说明有转录激活活性，如果不能生长则说明没有转录激活活性。同时，将阳性酵母菌落进行X-gal显色反应，检测LacZ报告基因表达情况，显蓝色的为阳性，说明具有转录激活功能，而不显蓝色的为阴性，说明不具有转录激活功能。具体的操作步骤如下：在培养皿中用5mL Z buffer/X-gal溶液浸湿一张无菌滤纸，挑取新鲜培养的酵母单菌落至另一张无菌滤纸上，于液氮中冷冻15秒后，置于室温融化。小心将带酵母的滤纸放到浸湿的滤纸上，置于30℃培养箱中，8小时内菌落出现蓝色的为阳性，说明具有转录激活功能。而不显蓝色的为阴性，说明不具有转录激活功能。实验结果见图4，图4说明GhDRP1全长蛋白具有转录激活活性，而截短后的蛋白片段都不具有转录激活活性。Construct pGBKT7-GhDRP1, pGBKT7-GhDRP1(1-354), pGBKT7-GhDRP1(355-1050), pGBKT7-GhDRP1(355-1134) and pGBKT7-GhDRP1(355-1254) vectors, transform yeast cells Y187 respectively, and detect The obtained positive yeast colony is streaked on the SD/-Ade/-Trp medium, if it can grow, it means that it has transcriptional activation activity, if it cannot grow, it means that it has no transcriptional activation activity. At the same time, the positive yeast colonies were subjected to X-gal color reaction to detect the expression of the LacZ reporter gene. The blue color was positive, indicating that it had the transcription activation function, and the blue color was negative, indicating that it did not have the transcription activation function. The specific operation steps are as follows: wet a piece of sterile filter paper with 5mL Z buffer/X-gal solution in a petri dish, pick a single colony of freshly cultured yeast onto another piece of sterile filter paper, freeze in liquid nitrogen for 15 Seconds later, thaw at room temperature. Carefully place the filter paper with yeast on the soaked filter paper, and place it in a 30°C incubator. If the colony appears blue within 8 hours, it is positive, indicating that it has the function of transcription activation. Those that do not show blue are negative, indicating that they do not have the function of transcriptional activation. The experimental results are shown in Figure 4, which shows that the full-length GhDRP1 protein has transcriptional activation activity, while the truncated protein fragments do not have transcriptional activation activity.
5.GhDRP1过量表达的转基因拟南芥表型分析5. Phenotype analysis of transgenic Arabidopsis overexpressed with GhDRP1
构建35S启动子驱动的pBI-GhDRP1过量表达载体，转化拟南芥，获得转基因植株。提取拟南芥植株的基因组DNA，用PCR技术检测鉴定阳性植株。在mRNA水平上检测GhDRP1在过量表达拟南芥植株中的表达水平，具体方法为分别提取了GhDRP1过量表达的转基因拟南芥植株的总RNA，逆转录为cDNA，以拟南芥的Actin2基因为内参，进行定量表达分析。然后，选择不同表达量的转基因株系进行表型分析，结果见图5和图6。The pBI-GhDRP1 overexpression vector driven by 35S promoter was constructed and transformed into Arabidopsis thaliana to obtain transgenic plants. Genomic DNA of Arabidopsis plants was extracted, and PCR technology was used to detect and identify positive plants. The expression level of GhDRP1 in the overexpressed Arabidopsis plants was detected at the mRNA level. The specific method was to extract the total RNA of the transgenic Arabidopsis plants overexpressed with GhDRP1, reverse transcribe it into cDNA, and use the Actin2 gene of Arabidopsis as Internal reference for quantitative expression analysis. Then, transgenic lines with different expression levels were selected for phenotypic analysis, and the results are shown in Fig. 5 and Fig. 6 .
6.GhDRP1过量表达(OE)和RNA干扰(RNAi)转基因棉花表型分析6. Phenotype analysis of GhDRP1 overexpression (OE) and RNA interference (RNAi) transgenic cotton
构建35S启动子驱动的pBI-GhDRP1载体和GhDRP1自身启动子驱动的RNAi载体，分别转化农杆菌LBA4404。利用农杆菌浸染棉花下胚轴外植体，共培养2天后，将下胚轴转移到选择培养基上培养，诱导并筛选到转化愈伤组织。将该愈伤组织经过8–10个月的继代培养后，诱导分化出胚性愈伤组织和体细胞胚，体细胞胚进而萌发再生为转基因棉花幼苗。将棉花幼苗移栽至土中，生长发育至开花结实。提取棉花基因组DNA，利用PCR技术检测鉴定转基因棉花植株。提取棉花叶片的RNA，利用定量RT-PCR技术分析转基因棉花中GhDRP1基因表达水平。然后，选择不同表达水平的转基因棉花株系进行表型分析和生理指标测定，见图7，结果显示在干旱处理后GhDRP1OE植株中丙二醛(MDA)和双氧水(H2O2)含量显著高于对照品种晋棉13号(J13)，而GhDRP1RNAi转基因棉花植株中MDA和H2O2含量显著低于对照品种。相反，GhDRP1RNAi植株中超氧化物歧化酶(SOD)和过氧化物酶(POD)活性显著升高，而GhDRP1过量表达植株中SOD和POD酶活性显著降低。这些结果表明过量表达GhDRP1减弱了棉花的抗旱性，而抑制GhDRP1基因表达能够提高棉花的抗旱性。The 35S promoter-driven pBI-GhDRP1 vector and the GhDRP1 self-promoter-driven RNAi vector were constructed and transformed into Agrobacterium LBA4404 respectively. Cotton hypocotyl explants were impregnated with Agrobacterium, and after co-cultivation for 2 days, the hypocotyls were transferred to the selection medium for culture, and the transformed callus was induced and screened. After the callus was subcultured for 8-10 months, embryogenic callus and somatic embryos were induced to differentiate, and the somatic embryos germinated and regenerated into transgenic cotton seedlings. The cotton seedlings are transplanted into the soil, and grow and develop until flowering and fruiting. Cotton genomic DNA was extracted, and PCR technology was used to detect and identify transgenic cotton plants. RNA was extracted from cotton leaves, and the expression level of GhDRP1 gene in transgenic cotton was analyzed by quantitative RT-PCR. Then, transgenic cotton lines with different expression levels were selected for phenotypic analysis and physiological index determination, as shown in Figure 7, the results show that the contents of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) in GhDRP1OE plants were significantly higher after drought treatment In the control variety Jinmian 13 (J13), the contents of MDA and H 2 O 2 in the GhDRP1RNAi transgenic cotton plants were significantly lower than those in the control variety. In contrast, the activities of superoxide dismutase (SOD) and peroxidase (POD) were significantly increased in GhDRP1 RNAi plants, while the activities of SOD and POD enzymes were significantly decreased in GhDRP1 overexpression plants. These results indicated that overexpression of GhDRP1 weakened the drought resistance of cotton, while suppressing GhDRP1 gene expression could improve the drought resistance of cotton.
说明书核苷酸和氨基酸序列表Instructions Nucleotide and Amino Acid Sequence Listing
＜110＞ 华中师范大学＜110＞ Central China Normal University
＜120＞ 棉花抗旱相关基因GhDRP1鉴定及应用＜120＞ Identification and application of drought resistance related gene GhDRP1 in cotton
＜160＞ 3＜160＞ 3
＜210＞ 1<210> 1
＜211＞ 1873bp<211> 1873bp
＜212＞ DNA<212> DNA
＜213＞ 棉花 (Gossypium hirsutum)＜213＞ Cotton (Gossypium hirsutum)
＜400＞ 1＜400＞ 1
ATGGCGGAGA TCTGTTACGG AGTTGTGAGC GAAGGCGAAG CATCAATACC GTGCGAGCCG 60ATGGCGGAGA TCTGTTACGG AGTTGTGAGC GAAGGCGAAG CATCAATACC GTGCGAGCCG 60
AGTTCACGTG CAGCAAGGAG GCGTAGGATG GAGATTAGAC GAATTAAAAT CGTCGACGTG 120 AGTTCACGTG CAGCAAGGAG GCGTAGGATG GAGATTAGAC GAATTAAAAT CGTCGACGTG 120
GCTCCATCTG AAGCTGACAG CGGCCGGAAA CGCAAGAACC TGCAAGCTTA CGGAGCATCA 180 GCTCCATCTG AAGCTGACAG CGGCCGGAAA CGCAAGAACC TGCAAGCTTA CGGAGCATCA 180
TTTTCTCTGG ACTGTGAAAA CGCTGTAGAA AATTGTGCCT CCGATGAGGA CGGAAAAAAG 240 TTTTCTCTGG ACTGTGAAAA CGCTGTAGAA AATTGTGCCT CCGATGAGGA CGGAAAAAAG 240
CGAACTGTTA AACCTAAAAA TGGAAGACTA AAAACGAAAG GAACAATAAT GAAAAGTAAC 300 CGAACTGTTA AACCTAAAAA TGGAAGACTA AAAACGAAAG GAACAATAAT GAAAAGTAAC 300
TCGAGTCCCT CGCTTTTGAT ACCTGAAATC GATTCAGAGT TACATCCTAA ATTCGGTGTC 360 TCGAGTCCCCT CGCTTTTGAT ACCTGAAATC GATTCAGAGT TACATCCTAA ATTCGGTGTC 360
GCTTCGGTTT GTGGTAGGAG AAGAGACATG GAAGACGCCG TCGCTATTCA TCCGTCTTTC 420 GCTTCGGTTT GTGGTAGGAG AAGAGACATG GAAGACGCCG TCGCTATTCA TCCGTCTTTC 420
CATCGTCAAG GCCAGGACTC TGCCGCCATT GGCTTCCACT ATTTTGGCGT CTATGATGGT 480 CATCGTCAAG GCCAGGACTC TGCCGCCATT GGCTTCCACT ATTTTGGCGT CTATGATGGT 480
CACGGTTGCT CTCATGTACG AGTCTGTAAA ATTTTAAAGT TAAAAATAGA TGTTCTGTTG 540 CACGGTTGCT CTCATGTACG AGTCTGTAAA ATTTTAAAGT TAAAAATAGA TGTTCTGTTG 540
GTTTTTTTCC ATCTCTGAAG AAATTGAATA TGCTTTTGGC GGTTTTGGTT ACTGTTTATT 600 GTTTTTTTCC ATCTCTGAAG AAATTGAATA TGCTTTTGGC GGTTTTGGTT ACTGTTTATT 600
AGGTGGCGAT GAGGTGCAGA GAGCGTTTAC ATGAGCTGGT GAAAGAAGAG TTGGCCAGTG 660 AGGTGGCGAT GAGGTGCAGA GAGCGTTTAC ATGAGCTGGT GAAAGAAGAG TTGGCCAGTG 660
AGGAGGAATG GAAAGGTGCT ATGGAGCGTA GCTTCACGCG CATGGATAAG GAAGTGATAA 720 AGGAGGAATG GAAAGGTGCT ATGGAGCGTA GCTTCACGCG CATGGATAAG GAAGTGATAA 720
AGTGGAACGA GAGCGTGGAT GGTGCTAATT GCCGATGCGA GTTACAGTCA CCCGAGTGTG 780 AGTGGAACGA GAGCGTGGAT GGTGCTAATT GCCGATGCGA GTTACAGTCA CCCGAGTGTG 780
ATACGGTTGG ATCTACAGCT GTTGTCGCTA TCGTAACGCC TGATAAAGTT GTTGTTGCTA 840 ATACGGTTGG ATCTACAGCT GTTGTCGCTA TCGTAACGCC TGATAAAGTT GTTGTTGCTA 840
ACTGTGGTGA TTCGAGAGCT GTTTTGTGTC GTAACGGCAG GCCTGTTCCT TTATCGTCCG 900 ACTGTGGTGA TTCGAGAGCT GTTTTGTGTC GTAACGGCAG GCCTGTTCCT TTATCGTCCG 900
ATCATAAGGT TAGCACTCGG TAGTTAGCTC TGTTATTTAA CGTAAAAACG TTCTAATTGT 960 ATCATAAGGT TAGCACTCGG TAGTTAGTC TGTTATTTAA CGTAAAAACG TTCTAATTGT 960
TTGAAGTTTG AGAAATCCTG CTAGTTTACA AGAAAATGTT TCTTAGCTGT CTCCGTATTT 1020 TTGAAGTTTG AGAAATCCTG CTAGTTTACA AGAAAATGTT TCTTAGCTGT CTCCGTATTT 1020
CTTTCTTGAT AACAAAAATT TGGTTCTTTT TCTATTTAAC CAAACAACAA GCTTTAAGGA 1080 CTTTCTTGAT AACAAAAATT TGGTTCTTTT TCTATTTAAC CAAACAACAA GCTTTAAGGA 1080
ATAAGATTTT CTCCGGAAAA TGTCTTCTTT TCCTTTTAAT TTCTATGCAG TCAAGCAGCT 1140 ATAAGATTTT CTCCGGAAAA TGTCTTCTTT TCCTTTTAAT TTCTATGCAG TCAAGCAGCT 1140
TAATGTATAA TATAGATTAG GTGCACTGCT CATACATATC ATGCGGAAAC AATGAGATAA 1200 TAATGTATAA TATAGATTAG GTGCACTGCT CATACATATC ATGCGGAAAC AATGAGATAA 1200
TCTTAATCTG ATGGTTTGGA CTTGTTTCGA TGTTTTAACG GCCAAAATTT GGAAGAAAGA 1260 TCTTAATCTG ATGGTTTGGA CTTGTTTCGA TGTTTTAACG GCCAAAATTT GGAAGAAAGA 1260
TTTGGAATTC TGTTTACTAT AATATGATAT TAAGTAAATA AACCTCCTTT CCCCCGTACG 1320 TTTGGAATTC TGTTTACTAT AATATGATAT TAAGTAAATA AACCTCCTTT CCCCCGTACG 1320
TGTGAACAGC CGGATCGTCC GGATGAGCTG AACCGGATCC AAGAAGCGGG AGGTCGGGTC 1380 TGTGAACAGC CGGATCGTCC GGATGAGCTG AACCGGATCC AAGAAGCGGG AGGTCGGGTC 1380
ATTTTCTGGG ATGGTCCTCG AGTTCTGGGA GTCCTCGCGA TGTCAAGAGC CATAGGTAAA 1440 ATTTTCTGGG ATGGTCCTCG AGTTCTGGGA GTCCTCGCGA TGTCAAGAGC CATAGGTAAA 1440
ATTTATTTAA AAGGCAGATC ATAACTTTCA TTCTATTTAA CATCAGGAGT ACGCCGCTAA 1500 ATTTATTTAA AAGGCAGATC ATAACTTTCA TTCTATTTAA CATCAGGAGT ACGCCGCTAA 1500
CTTGAAAAAC ATTGTCTACG CCACAGGTGA TAACTACTTG AAACCCTACG TGAGCTGTGA 1560 CTTGAAAAAC ATTGTCTACG CCACAGGTGA TAACTACTTG AAACCCTACG TGAGCTGTGA 1560
GCCGGAGGTT ACAGTAACGG ATCGAACGGC AGAGGACGAA TGTTTGATTC TGGCGAGTGA 1620 GCCGGAGGTT ACAGTAACGG ATCGAACGGC AGAGGACGAA TGTTTGATTC TGGCGAGTGA 1620
CGGTTTATGG GACGTGGTAT CAAATGATAC TGCATGCGGG GTGGCGCGCA TGTGTTTGAG 1680 CGGTTTATGG GACGTGGTAT CAAATGATAC TGCATGCGGG GTGGCGCGCA TGTGTTTGAG 1680
GGGAAAGTGT GATGTACAGG CGCCGCTATT GTCACCGGAA GGAGAGGCGG TTGTAGGGTC 1740 GGGAAAGTGT GATGTACAGG CGCCGCTATT GTCACCGGAA GGAGAGGCGG TTGTAGGGTC 1740
GATGATGGGA GGAGGAGAGA TCCCGGACAA GGCGTGCGCT GATGCGTCCA TGTTGTTGAC 1800 GATGATGGGA GGAGGAGAGA TCCCGGACAA GGCGTGCGCT GATGCGTCCA TGTTGTTGAC 1800
AAAGCTGGCG TTGGCCAGGC ATAGTACGGA CAATGTTAGC GTAGTCGTGG TGGATCTAAG 1860 AAAGCTGGCG TTGGCCAGGC ATAGTACGGA CAATGTTAGC GTAGTCGTGG TGGATCTAAG 1860
GAGAGCCACG TAA 1873 GAGAGCCACG TAA 1873
＜210＞ 2<210> 2
＜211＞ 1254bp<211> 1254bp
＜212＞ DNA<212> DNA
＜213＞ 棉花 (Gossypium hirsutum)＜213＞ Cotton (Gossypium hirsutum)
＜400＞ 1＜400＞ 1
ATGGCGGAGA TCTGTTACGG AGTTGTGAGC GAAGGCGAAG CATCAATACC GTGCGAGCCG 60ATGGCGGAGA TCTGTTACGG AGTTGTGAGC GAAGGCGAAG CATCAATACC GTGCGAGCCG 60
AGTTCACGTG CAGCAAGGAG GCGTAGGATG GAGATTAGAC GAATTAAAAT CGTCGACGTG 120 AGTTCACGTG CAGCAAGGAG GCGTAGGATG GAGATTAGAC GAATTAAAAT CGTCGACGTG 120
GCTCCATCTG AAGCTGACAG CGGCCGGAAA CGCAAGAACC TGCAAGCTTA CGGAGCATCA 180 GCTCCATCTG AAGCTGACAG CGGCCGGAAA CGCAAGAACC TGCAAGCTTA CGGAGCATCA 180
TTTTCTCTGG ACTGTGAAAA CGCTGTAGAA AATTGTGCCT CCGATGAGGA CGGAAAAAAG 240 TTTTCTCTGG ACTGTGAAAA CGCTGTAGAA AATTGTGCCT CCGATGAGGA CGGAAAAAAG 240
CGAACTGTTA AACCTAAAAA TGGAAGACTA AAAACGAAAG GAACAATAAT GAAAAGTAAC 300 CGAACTGTTA AACCTAAAAA TGGAAGACTA AAAACGAAAG GAACAATAAT GAAAAGTAAC 300
TCGAGTCCCT CGCTTTTGAT ACCTGAAATC GATTCAGAGT TACATCCTAA ATTCGGTGTC 360 TCGAGTCCCCT CGCTTTTGAT ACCTGAAATC GATTCAGAGT TACATCCTAA ATTCGGTGTC 360
GCTTCGGTTT GTGGTAGGAG AAGAGACATG GAAGACGCCG TCGCTATTCA TCCGTCTTTC 420 GCTTCGGTTT GTGGTAGGAG AAGAGACATG GAAGACGCCG TCGCTATTCA TCCGTCTTTC 420
CATCGTCAAG GCCAGGACTC TGCCGCCATT GGCTTCCACT ATTTTGGCGT CTATGATGGT 480 CATCGTCAAG GCCAGGACTC TGCCGCCATT GGCTTCCACT ATTTTGGCGT CTATGATGGT 480
CACGGTTGCT CTCATGTGGC GATGAGGTGC AGAGAGCGTT TACATGAGCT GGTGAAAGAA 540 CACGGTTGCT CTCATGTGGC GATGAGGTGC AGAGAGCGTT TACATGAGCT GGTGAAAGAA 540
GAGTTGGCCA GTGAGGAGGA ATGGAAAGGT GCTATGGAGC GTAGCTTCAC GCGCATGGAT 600 GAGTTGGCCA GTGAGGAGGA ATGGAAAGGT GCTATGGAGC GTAGCTTCAC GCGCATGGAT 600
AAGGAAGTGA TAAAGTGGAA CGAGAGCGTG GATGGTGCTA ATTGCCGATG CGAGTTACAG 660 AAGGAAGTGA TAAAGTGGAA CGAGAGCGTG GATGGTGCTA ATTGCCGATG CGAGTTACAG 660
TCACCCGAGT GTGATACGGT TGGATCTACA GCTGTTGTCG CTATCGTAAC GCCTGATAAA 720 TCACCCGAGT GTGATACGGT TGGATCTACA GCTGTTGTCG CTATCGTAAC GCCTGATAAA 720
GTTGTTGTTG CTAACTGTGG TGATTCGAGA GCTGTTTTGT GTCGTAACGG CAGGCCTGTT 780 GTTGTTGTTG CTAACTGTGG TGATTCGAGA GCTGTTTTGT GTCGTAACGG CAGGCCTGTT 780
CCTTTATCGT CCGATCATAA GCCGGATCGT CCGGATGAGC TGAACCGGAT CCAAGAAGCG 840 CCTTTATCGT CCGATCATAA GCCGGATCGT CCGGATGAGC TGAACCGGAT CCAAGAAGCG 840
GGAGGTCGGG TCATTTTCTG GGATGGTCCT CGAGTTCTGG GAGTCCTCGC GATGTCAAGA 900 GGAGGTCGGG TCATTTTCTG GGATGGTCCT CGAGTTCTGG GAGTCCTCGC GATGTCAAGA 900
GCCATAGGTG ATAACTACTT GAAACCCTAC GTGAGCTGTG AGCCGGAGGT TACAGTAACG 960 GCCATAGGTG ATAACTACTT GAAACCCTAC GTGAGCTGTG AGCCGGAGGT TACAGTAACG 960
GATCGAACGG CAGAGGACGA ATGTTTGATT CTGGCGAGTG ACGGTTTATG GGACGTGGTA 1020 GATCGAACGG CAGAGGACGA ATGTTTGATT CTGGCGAGTG ACGGTTTATG GGACGTGGTA 1020
TCAAATGATA CTGCATGCGG GGTGGCGCGC ATGTGTTTGA GGGGAAAGTG TGATGTACAG 1080 TCAAATGATA CTGCATGCGG GGTGGCGCGC ATGTGTTTGA GGGGAAAGTG TGATGTACAG 1080
GCGCCGCTAT TGTCACCGGA AGGAGAGGCG GTTGTAGGGT CGATGATGGG AGGAGGAGAG 1140 GCGCCGCTAT TGTCACCGGA AGGAGAGGCG GTTGTAGGGT CGATGATGGG AGGAGGAGAG 1140
ATCCCGGACA AGGCGTGCGC TGATGCGTCC ATGTTGTTGA CAAAGCTGGC GTTGGCCAGG 1200 ATCCCGGACA AGGCGTGCGC TGATGCGTCC ATGTTGTTGA CAAAGCTGGC GTTGGCCAGG 1200
CATAGTACGG ACAATGTTAG CGTAGTCGTG GTGGATCTAA GGAGAGCCAC GTAA 1254 CATAGTACGG ACAATGTTAG CGTAGTCGTG GTGGATCTAA GGAGAGCCAC GTAA 1254
＜210＞ 3<210> 3
＜211＞ 417<211> 417
＜212＞ PRT＜212＞PRT
＜213＞ 棉花 (Gossypium hirsutum)＜213＞ Cotton (Gossypium hirsutum)
＜400＞ 1＜400＞ 1
Met Ala Glu Ile Cys Tyr Gly Val Val Ser Glu Gly Glu Ala SerMet Ala Glu Ile Cys Tyr Gly Val Val Ser Glu Gly Glu Ala Ser
1 5 10 151 5 10 15
Ile Pro Cys Glu Pro Ser Ser Arg Ala Ala Arg Arg Arg Arg MetIle Pro Cys Glu Pro Ser Ser Arg Ala Ala Arg Arg Arg Arg Met
20 25 30 20 25 30
Glu Ile Arg Arg Ile Lys Ile Val Asp Val Ala Pro Ser Glu AlaGlu Ile Arg Arg Ile Lys Ile Val Asp Val Ala Pro Ser Glu Ala
35 40 45 35 40 45
Asp Ser Gly Arg Lys Arg Lys Asn Leu Gln Ala Tyr Gly Ala SerAsp Ser Gly Arg Lys Arg Lys Asn Leu Gln Ala Tyr Gly Ala Ser
50 55 60 50 55 60
Phe Ser Leu Asp Cys Glu Asn Ala Val Glu Asn Cys Ala Ser AspPhe Ser Leu Asp Cys Glu Asn Ala Val Glu Asn Cys Ala Ser Asp
65 70 75 65 70 75
Glu Asp Gly Lys Lys Arg Thr Val Lys Pro Lys Asn Gly Arg LeuGlu Asp Gly Lys Lys Arg Thr Val Lys Pro Lys Asn Gly Arg Leu
80 85 90 80 85 90
Lys Thr Lys Gly Thr Ile Met Lys Ser Asn Ser Ser Pro Ser LeuLys Thr Lys Gly Thr Ile Met Lys Ser Asn Ser Ser Pro Ser Leu
95 100 105 95 100 105
Leu Ile Pro Glu Ile Asp Ser Glu Leu His Pro Lys Phe Gly ValLeu Ile Pro Glu Ile Asp Ser Glu Leu His Pro Lys Phe Gly Val
110 115 120 110 115 120
Ala Ser Val Cys Gly Arg Arg Arg Asp Met Glu Asp Ala Val AlaAla Ser Val Cys Gly Arg Arg Arg Asp Met Glu Asp Ala Val Ala
125 130 135 125 130 135
Ile His Pro Ser Phe His Arg Gln Gly Gln Asp Ser Ala Ala IleIle His Pro Ser Phe His Arg Gln Gly Gln Asp Ser Ala Ala Ile
140 145 150 140 145 150
Gly Phe His Tyr Phe Gly Val Tyr Asp Gly His Gly Cys Ser HisGly Phe His Tyr Phe Gly Val Tyr Asp Gly His Gly Cys Ser His
155 160 165 155 160 165
Val Ala Met Arg Cys Arg Glu Arg Leu His Glu Leu Val Lys GluVal Ala Met Arg Cys Arg Glu Arg Leu His Glu Leu Val Lys Glu
170 175 180 170 175 180
Glu Leu Ala Ser Glu Glu Glu Trp Lys Gly Ala Met Glu Arg SerGlu Leu Ala Ser Glu Glu Glu Trp Lys Gly Ala Met Glu Arg Ser
185 190 195 185 190 195
Phe Thr Arg Met Asp Lys Glu Val Ile Lys Trp Asn Glu Ser ValPhe Thr Arg Met Asp Lys Glu Val Ile Lys Trp Asn Glu Ser Val
200 205 210 200 205 210
Asp Gly Ala Asn Cys Arg Cys Glu Leu Gln Ser Pro Glu Cys AspAsp Gly Ala Asn Cys Arg Cys Glu Leu Gln Ser Pro Glu Cys Asp
215 220 225 215 220 225
Thr Val Gly Ser Thr Ala Val Val Ala Ile Val Thr Pro Asp LysThr Val Gly Ser Thr Ala Val Val Ala Ile Val Thr Pro Asp Lys
230 235 240 230 235 240
Val Val Val Ala Asn Cys Gly Asp Ser Arg Ala Val Leu Cys ArgVal Val Val Ala Asn Cys Gly Asp Ser Arg Ala Val Leu Cys Arg
245 250 255 245 250 255
Asn Gly Arg Pro Val Pro Leu Ser Ser Asp His Lys Pro Asp ArgAsn Gly Arg Pro Val Pro Leu Ser Ser Asp His Lys Pro Asp Arg
260 265 270 260 265 270
Pro Asp Glu Leu Asn Arg Ile Gln Glu Ala Gly Gly Arg Val IlePro Asp Glu Leu Asn Arg Ile Gln Glu Ala Gly Gly Arg Val Ile
275 280 285 275 280 285
Phe Trp Asp Gly Pro Arg Val Leu Gly Val Leu Ala Met Ser ArgPhe Trp Asp Gly Pro Arg Val Leu Gly Val Leu Ala Met Ser Arg
290 295 300 290 295 300
Ala Ile Gly Asp Asn Tyr Leu Lys Pro Tyr Val Ser Cys Glu ProAla Ile Gly Asp Asn Tyr Leu Lys Pro Tyr Val Ser Cys Glu Pro
305 310 315 305 310 315
Glu Val Thr Val Thr Asp Arg Thr Ala Glu Asp Glu Cys Leu IleGlu Val Thr Val Thr Asp Arg Thr Ala Glu Asp Glu Cys Leu Ile
320 325 330 320 325 330
Leu Ala Ser Asp Gly Leu Trp Asp Val Val Ser Asn Asp Thr AlaLeu Ala Ser Asp Gly Leu Trp Asp Val Val Ser Asn Asp Thr Ala
335 340 345 335 340 345
Cys Gly Val Ala Arg Met Cys Leu Arg Gly Lys Cys Asp Val GlnCys Gly Val Ala Arg Met Cys Leu Arg Gly Lys Cys Asp Val Gln
350 355 360 350 355 360
Ala Pro Leu Leu Ser Pro Glu Gly Glu Ala Val Val Gly Ser MetAla Pro Leu Leu Ser Pro Glu Gly Glu Ala Val Val Gly Ser Met
365 370 375 365 370 375
Met Gly Gly Gly Glu Ile Pro Asp Lys Ala Cys Ala Asp Ala SerMet Gly Gly Gly Glu Ile Pro Asp Lys Ala Cys Ala Asp Ala Ser
380 385 390 380 385 390
Met Leu Leu Thr Lys Leu Ala Leu Ala Arg His Ser Thr Asp AsnMet Leu Leu Thr Lys Leu Ala Leu Ala Arg His Ser Thr Asp Asn
395 400 405 395 400 405
Val Ser Val Val Val Val Asp Leu Arg Arg Ala ThrVal Ser Val Val Val Val Asp Leu Arg Arg Ala Thr
410 415 417 410 415 417
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110791523A (en) * | 2019-12-13 | 2020-02-14 | 南京农业大学 | A cotton drought resistance related gene GhRCHY1 and its application |
| CN110872598A (en) * | 2019-12-13 | 2020-03-10 | 南京农业大学 | A kind of cotton drought resistance related gene GhDT1 and its application |
| CN112048490A (en) * | 2020-09-18 | 2020-12-08 | 中国农业大学 | Cotton silk/threonine protein phosphatase GhTPOPP 6 and coding gene and application thereof |
| CN112342225A (en) * | 2020-12-10 | 2021-02-09 | 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） | Drought-resistant gene and application thereof |
| CN114634941A (en) * | 2022-05-16 | 2022-06-17 | 中国农业科学院生物技术研究所 | Upland cotton GhPP2Cs gene and application thereof in plant dwarfing |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020023281A1 (en) * | 2000-01-27 | 2002-02-21 | Jorn Gorlach | Expressed sequences of arabidopsis thaliana |
| CN102134574A (en) * | 2010-01-21 | 2011-07-27 | 湖南大学 | Arabidopsis thaliana atpp2ca2 gene and application thereof |
| WO2012085862A2 (en) * | 2010-12-22 | 2012-06-28 | Evogene Ltd. | Isolated polynucleotides and polypeptides, and methods of using same for increasing abiotic stress tolerance, yield, growth rate, vigor, biomass, oil content, and/or nitrogen use efficiency of plants |
| CN105294847A (en) * | 2015-12-02 | 2016-02-03 | 中国农业科学院生物技术研究所 | Stress tolerance-related protein of plants and encoding gene and application of stress tolerance-related protein |
-
2016
- 2016-11-28 CN CN201611065602.0A patent/CN106520798A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020023281A1 (en) * | 2000-01-27 | 2002-02-21 | Jorn Gorlach | Expressed sequences of arabidopsis thaliana |
| CN102134574A (en) * | 2010-01-21 | 2011-07-27 | 湖南大学 | Arabidopsis thaliana atpp2ca2 gene and application thereof |
| WO2012085862A2 (en) * | 2010-12-22 | 2012-06-28 | Evogene Ltd. | Isolated polynucleotides and polypeptides, and methods of using same for increasing abiotic stress tolerance, yield, growth rate, vigor, biomass, oil content, and/or nitrogen use efficiency of plants |
| CN105294847A (en) * | 2015-12-02 | 2016-02-03 | 中国农业科学院生物技术研究所 | Stress tolerance-related protein of plants and encoding gene and application of stress tolerance-related protein |
Non-Patent Citations (2)
| Title |
|---|
| XM_016833616.1: "PREDICTED: Gossypium hirsutum probable protein phosphatase 2C 24 (LOC107906580),mRNA", 《GENBANK》 * |
| XP_016689105.1: "PREDICTED: probable protein phosphatase 2C 24 [Gossypium hirsutum]", 《GENPEPT》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110791523A (en) * | 2019-12-13 | 2020-02-14 | 南京农业大学 | A cotton drought resistance related gene GhRCHY1 and its application |
| CN110872598A (en) * | 2019-12-13 | 2020-03-10 | 南京农业大学 | A kind of cotton drought resistance related gene GhDT1 and its application |
| CN110791523B (en) * | 2019-12-13 | 2022-05-10 | 南京农业大学 | Cotton drought-resistant related gene GhRCHY1 and application thereof |
| CN112048490A (en) * | 2020-09-18 | 2020-12-08 | 中国农业大学 | Cotton silk/threonine protein phosphatase GhTPOPP 6 and coding gene and application thereof |
| CN112342225A (en) * | 2020-12-10 | 2021-02-09 | 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） | Drought-resistant gene and application thereof |
| CN114634941A (en) * | 2022-05-16 | 2022-06-17 | 中国农业科学院生物技术研究所 | Upland cotton GhPP2Cs gene and application thereof in plant dwarfing |
| CN114634941B (en) * | 2022-05-16 | 2022-08-12 | 中国农业科学院生物技术研究所 | Upland cotton GhPP2Cs gene and application thereof in plant dwarfing |
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